Neighbor effects in the mutation of ochre triplets in the T 4 rII gene

In 15 sites in the T₄rII gene, mutation from the ochre (UAA) codon to amber (UAG), opal (UGA) and the wild-type was measured with and without 2-aminopurine treatment. It is shown that a particular base pair in the DNA may show variable mutability, depending on its nearest neighbors. Also, similar base pairs at different sites in the gene can vary in their mutability despite the fact that they are flanked by similar neighbors.     

Novel rII duplications in bacteriophage T4.

The properties of two rII complementation heterozygotes (D5B and D7A) of bacteriophage T4 are described. These strains are characterized by their stability, each forming less than 10-3 r segregants among their viable progeny, and by their segregation of only one of the two parental types. No increase in r progeny was found on crossing D7A or D5B with T4r+, indicating that the duplications in these strains are not separated by an essential region of the phage genome. Both D5B and D7A from h-2+/h-4+ heterozygotes at frequencies similar to T4r+, suggesting that the duplicated regions in these strains are short. The progeny of these h-2+/h-4+ heterozygotes retain heterozygosity for rII but not for h: therefore, D5B and D7A are not stabilized terminal redundancy complementation heterozygotes. We conclude that D5B and D7A contain very short tandem duplications and we present structures consistent with the observed characteristics of these phages.     

Marker Effects on Reversion of T4rII Mutants

The frequencies of 2-aminopurine- and 5-bromouracil-induced A:T leads to G:C transitions were compared at nonsense sites throughout the rII region of bacteriophage T4. These frequencies are influenced both by adjacent base pairs within the nonsense codons and by extracodonic factors. Following 2AP treatment, they are high in amber (UAG) and lower in opal (UGA) codons than in allelic ochre (UAA) codons. In general, 5BU-induced transitions are more frequent in both amber and opal codons than in the allelic ochre codons. 2AP- and 5BU-induced transition frequencies in the first and third positions of opal codons are correlated with those in the corresponding positions of the allelic ochre codons. Similarly, the frequencies of 2AP-induced transition in the first and second positions of amber codons and their ochre alleles are correlated. However, there is little correlation between the frequencies of 5BU-induced transitions in the first and second positions of allelic amber and ochre codons.     

Replication and Recombination in Ligase-Deficient rII Bacteriophage T4D

Deoxyribonucleic acid replication and genetic recombination were investigated after infection of Escherichia coli with ligase-deficient rII bacteriophage T4D. The major observations are: (i) deoxyribonucleic acid synthesis is discontinuous, (ii) the discontinuities are more slowly repaired than in wild-type infection, (iii) host ligase is required for viability, and (iv) genetic recombination is increased.     

Parent-to-Progeny Transfer and Recombination of T4rII Bacteriophage

Transfer of parental, light (not substituted with 5-bromodeoxyuridine) (32)P-deoxyribonucleic acid (DNA) from rII(-) mutants of T4 bacteriophage to heavy (5-bromodeoxyuridine-substituted) progeny in Escherichia coli B was less homogeneous than in wild phages. The net transfer was 5 to 20% of the value for wild T4 phage, and the parental contribution per progeny DNA molecule amounted to 7 to 100% of the genome. Three classes could be distinguished, based on the density distribution of parental label in CsCl analysis of the progeny phages. "Far recombined" phages contain parental material only in semiconservatively replicated subunits covalently attached to progeny DNA, amounting to 5 to 10% parental contribution per genome. "Intermediate recombinants" contain, aside from conventional recombinant DNA, parental DNA banding at the original, light density. This DNA may be unattached to heavy progeny DNA or attached by weak bonds which are very sensitive to shearing during the extraction procedure. The parental contribution is 10 to 50% per progeny DNA molecule in this class. "Conservative" phages band close to the parental, light density in CsCl; their DNA is purely light. When the parental phage is labeled with both (3)H-leucine (capsid) and (32)P (DNA), the specific activity of (3)H/(32)P in the "conservative progeny" is 10 to 40% of that in the parental, showing that at least some of the (32)P in this area belongs to phages with parental DNA as the sole DNA component inside an unlabeled capsid, i.e., parental DNA which has been injected into the host and matured in a new capsid without replication or recombination. This phenomenon occurs to about the same extent in both single and multiple infection.     

Neighbour and temperature effects on base-analogue-induced mutation in phage T4

Summary The effect of neighbouring base pairs and of temperature on mutation frequencies were measured at nonsense sites in the T4rII region. 2AP-induced ATGC transition frequencies are insensitive to nearest-neighbour effects, while 5BU-induced ones are promoted by GC neighbours on the 5 side. The effect of temperature on 2AP-and 5BU-induced mutation frequencies shows no simple dependence on nearest neighbours. These results are incompatible with a unitary mechanism as explanation for the effects of nearest neighbours and temperature on baseanalogue-induced mutagenesis.     

Analysis of expression of the rII gene function of bacteriophage T4.

Temperature-sensitive (ts) mutants of the T4 phage rII gene were islated and used in temperature shift experiments that revelaed two different expressions for the normal rII (rII+) gene function in vivo: (i) an early expression (0 to 12 min postinfection at 30 C) that prevents restriction of T4 growth in Escherichia coli hosts lysogenic for gamma phage, and (ii) a later expression (12 to 18 min postinfection at 30 C) that results in restriction of T4 growth when the phage DNA ligase (gene 30) is missing. The earlier expression appeared to coincide with the period of synthesis of the protein product of the T4 rIIA cistron, whereas the later expression occurred after rIIA protein synthesis had stopped. The synthesis of the protein product of the rIIB cistron continues for several minutes after rIIA protein synthesis ceases (O'Farrell and Gold, 1973). The two rII+ gene expressions might require different molar ratios of the rIIA and rIIB proteins. It is possible that the separate expressions of rII+ gene function are manifestations of different associations between the two rII proteins and other T4-induced proteins that are synthesized or activated at different times after phage infection.     

Nonsense Mutants in the rII A Cistron of Bacteriophage T4

After in vitro treatment of bacteriophage T4 with hydroxylamine (HA), 54 nonsense mutants in the rII A cistron were isolated. These mutants were characterized by growth on suppressor strains of Escherichia coli, and the mutational sites were mapped in the rII A cistron. Twenty-five (9 sites) were amber (UAG), 20 (6 sites) were opal (UGA), and 9 (6 sites) were ochre (UAA). Mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably involving mutations occurring in adjacent nucleotides. Based on the specificity of HA mutagenesis (GC → AT), the amino acid codons in which the mutations occurred have been inferred. It is suggested that the three amber-opal pairs arose in tryptophan codons (UGG) and the six ochre mutants arose in glutamine codons (CAA). The six unpaired ambers and the three unpaired opals have been tentatively assigned to glutamine codons (CAG) and arginine codons (CGA), respectively, in the wild-type phage.     

Genotypic reversion by methylene Blue: The orientation of guanine-hydroxymethylcytosine at mutated sites in rII mutants of phage T4

Summary Methylene Blue (MB) induced reversion of rII mutants to which a GC base pair was previously assigned is investigated, both in K₁₂() and in B bacteria. The rII mutants used are the same ones whose hydroxylamine (HA) induced reversion rates have previously been investigated by Levisohn (1967) in a similar series of experiments.The results show a relationship between MB-induced and HA-induced reversion patterns which can be interpreted on the assumptions: 1. Both MB and HA produce transitions, but MB acts on Guanine (in agreement with Simon and van Vunakis 1962), while HA acts on Cytocine; 2. Only one injected DNA strand (genetic strand) transmits hereditary information to the progeny while the other strand (associated strand) is disintegrated soon after performing some early functions and its nucleotides are re-used in the formation of new DNA; 3. When the injected associated strand disintegrates, Cytocine bases altered by HA can be re-used, while Guanine bases altered by MB cannot.The assumption that only one strand of the injected DNA transmits hereditary information to the progeny (Simplex hypotheses) is also used in a new interpretation of the mutagenic-induced reversion pattern of lysozyme mutants. This new interpretation avoids a difficulty in the earlier interpretation proposed by Terzaghi, Streisinger and Stahl (1962) which assigned an AT base pair to a mutant strongly HA revertible, while none of the mutants to which a GC base pair had been assigned was HA revertible.The main advantage of the Simplex interpretations of mutagenic phenomena is that self-consistent base assignments to each mutant can be made following a series of rules which have no exceptions: 1. An early function mutant with a high HA reversion rate in non-permissive host can consistently be assigned an odd C in the genetic strand (if odd designates a base which, when replaced through a transition, causes reversion to wild type); 2. A mutant with high MB reversion rate can consistently be assigned an odd G in the genetic strand; 3. A few mutants which present both of these properties are assigned both an odd C and an odd G in a genetic strand triplet; 4. A high HA reversion rate in permissive host but not in non-permissive host is consistently indicative of an odd G with no odd C in the genetic strand triplet.This work was supported by research grant GM-12581 from the Division of General Medical Sciences of the National Institutes of Health, United States Public Health Service.     

UV-induced mutation in bacteriophage T4.

Two late gene am mutants of bacteriophage T4 that can be induced to revert by UV were crossed to a temperature-sensitive ligase mutant. In the double mutants, UV-induced reversion was eliminated at a semirestrictive temperature. When the single am mutants were irradiated and then allowed a single passage in a permissive host, the UV-induced reversion frequency was increased by 15- to 25-fold. This increased mutagenesis was also abolished by the presence of the ligase allele. When the UV-irradiated single am mutants multiply infected a permissive host, allowing multiplicity reactivation to occur, the induced reversion frequency was reduced similarly to the reduction in lethality. The mutagenesis that remained was again abolished by the presence of the ligase allele. It is concluded that UV induces mutations in phage T4 through the action of a pathway that includes polynucleotide ligase. The increase in mutation frequency after growth in a permissive host implies that mutagenesis can occur at more than one stage of the infection rather than only in an early stage before expression of the mutant genome. The process of multiplicity reactivation appears to be error-free since it overcomes lethal lesions without inducing new mutations.     

Construction and properties of recombinant plasmids containing the rII genes of bacteriophage T4.

Summary The EcoRI digestion products of phage T4 DNA have been examined using a phage DNA transformation assay. A 2.6x10⁶ Dalton fragment was found to contain the rII genes. This fragment was purified and then treated with HindIII endonuclease. The cleavage products were ligated to the vector plasmid pBR313 and viable recombinant plasmids recovered. A genetic assay was employed to demonstrate that the recombinants contained T4 DNA and to localize on the phage genetic map the EcoRI and HindIII sites cleaved during the construction of the plasmids. Preliminary characterization suggests that a fragment covering the beginning of the rIIA gene possibly contains a promotor which is active in uninfected cells.Abbreviations used Ap ampicillin - Tc tetracycline - Mdal 10⁶ Daltons - bp base pairs     

Spectrum of spontaneous frameshift mutations. Sequences of bacteriophage T4 rII gene frameshifts

The DNA sequences of 185 independent spontaneous frameshift mutations in the rIIB gene of bacteriophage T4 are described. Approximately half of the frameshifts, including those at hot spot sites, are fully consistent with classical proposals that frameshift mutations are produced by a mechanism involving the misaligned pairing of repeated DNA sequences. However, the remaining frameshifts are inconsistent with this model. Correlations between the positions of two base-pair frameshifts and the bases of DNA hairpins suggest that local DNA topology might influence frameshift mutation. Warm spots for larger deletions share the property of having endpoints adjacent to DNA sequences whose complementarity to sequences a few base-pairs away suggest that non-classical DNA misalignments may participate in deletion mutation. A model for duplication mutation as a consequence of strand displacement synthesis is discussed. In all, 15 frameshifts were complex combinations of frameshifts and base substitutions. Three of these were identical, and have extended homology to a sequence 256 base-pairs away that is likely to participate in the mutational event; the remainder are unique combinations of frameshifts and transversions. The frequency and diversity of complex mutants suggest a challenge to the assumption that the molecular evolution of DNA must depend primarily upon the accumulation of single nucleotide changes.     

Defective Deoxyribonucleic Acid Replication of T4rII Bacteriophage in Lambda-Lysogenic Host Cells

Sedimentation of the replicative deoxyribonucleic acid through alkaline sucrose gradients showed that rII single chains reached the half-mature size at a time when wild-type molecules formed long chains (dimers and trimers of genome size). Long rII single chains could be observed on substitution of tris(hydroxymethyl)aminomethane buffer for Na⁺K⁺ phosphate in the growth medium.